This proposal will define signaling mechanisms associated with the extreme anterior domain (EAD) and face formation using the frogs Xenopus laevis and X. tropicalis as models. The EAD is a conserved embryonic region where ectoderm and endoderm are juxtaposed, that develops into the mouth, anterior pituitary and nostrils. In the previous funding period, we made several novel findings. (1) The EAD is an organizing center necessary for cranial neural crest (CNC) development, using the Kinin-Kallikrein pathway and nitric oxide (NO); (2) Incoming CNC induces the EAD to undergo convergent extension, a novel step in mouth formation, using the Wnt/PCP pathway; (3) Wnt antagonists frzb1+crescent that are localized in the EAD act globally, in other facial regions. Our data give insight into the earliest stages of facial development and suggest two hypotheses with high impact that will be addressed in this proposal. Wnt antagonists from the EAD regulate frontonasal prominence (FNP) and first arch CNC development. The EAD signals through Bradykinin and nitric oxide to regulate CNC migration, in a concentration-dependent manner. There are two Aims. The first Aim will delineate the role of Frzb1+Crescent derived from the EAD in neural crest development. The role(s) of EAD-derived Frzb1+Crescent in FNP and first arch CNC determination, migration, proliferation and survival will be assessed after local loss of function (LOF). X. tropicalis mutants will complement and extend analyses. Frzb1+Crescent will be assayed for sufficiency to direct neural crest development, using heterologous cells expressing Frzb1 or beads soaked in Frzb-IgG protein. Ability of Frzb1+Crescent to inhibit ?-catenin-mediated Wnt reporter activity in the FNP and first arch CNC will be assessed. Signaling pathways and candidate target genes modulated by frzb1+crescent will be determined. The second Aim will define the role of the EAD and the Kinin-Kallikrein pathway in modulating nitric oxide (NO) signaling and CNC migration. Cells in the developing facial region which produce NO will be identified, using the NO sensor DAF2 and NO-sensor nanotubes. A requirement for the EAD and EAD Kinin-Kallikrein factors in facial NO production will be assessed by extirpation and local LOF. cCNC cells will be assayed for ability to respond directly to Bdk peptides or NO in the embryo or in culture. Downstream effects of NO on target cells will be examined focusing on changes in signaling pathways and the cytoskeleton. The face is the defining feature of the individual human. Facial abnormalities are frequent (~1/700 births), resulting in physical and psychological disturbances. Much of the face may be impacted by reduced activity of the EAD organizer. The data obtained here will inform pre-natal diagnosis and correction, and are highly significant in the craniofacial field.